Micro fluidic structures

ABSTRACT

A micro fluidic system includes a substrate, and, provided on said substrate, at least one flow path interconnecting with functional means in which liquid samples can be treated by desired procedures. The flow paths are laid out to form a pattern for the transport of liquid samples to and from said functional means. These flow paths comprise a plurality of micro posts protruding upwards from said substrate, the spacing between the micro posts being small enough to induce a capillary action in a liquid sample applied anywhere within any of said flow paths, so as to force said liquid to move from where said liquid sample was applied.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/213,685, filed Aug. 19, 2011, which is a continuation of U.S. Pat.No. 8,025,854, issued Sep. 27, 2011, which claims the benefit of PCTPatent Application No. PCT/SE03/00919, filed on Jun. 4, 2003, whichclaims the benefit of Swedish Patent Application No. SE 0201738-2, filedon Jun. 7, 2002. The entirety of each of the above patent documents areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to micro fluidic structures, and inparticular to a micro structure defining a liquid flow system, whereincapillary action is utilized as the main driving force for liquidtransport through said structure.

The micro fluidic structure according to the invention is useful invarious fields of application such as miniaturized bioassays,preparatory steps for such assays, separation, electrophoresis,capillary chromatography, micro reaction cavity procedures, miniaturizedliquid communication units, biosensor flow cells and the like.

BACKGROUND OF THE INVENTION

Liquid transport through channels or structures on a micro scale hasimportant implications in a number of different technologies.

Controlled transport of fluids through micro channels has been achallenge, with the microstructure itself imparting difficulties notfound on a larger scale. The driving force utilized in most microchannel structures depends on electro-endosmosis, gravitational forces,external pressure or capillary migration.

Surface materials often have unbound electrons, polar moieties, or otherfeatures generating a surface charge or reactivity. Surfacecharacteristics often have a more pronounced impact on a micro scalesystem than on a larger structure. This is particularly true in microsystems where the fluid flow is driven by attractions between liquidsand the surface materials through which they are transported.

In a closed capillary the driving force is usually represented by theequation:

h=2σ_(gl) cos(θ_(c))/gρ

where h=the height of a fluid within a capillary tube; θ_(c)=the contactangle of the fluid with the capillary tube material.

If the contact angle of the capillary tube material, with respect to thefluid, is less than 90°, the material is considered hydrophilic. If thecontact angle of the tube material, with respect to the fluid, isgreater than 90°, the material is considered hydrophobic. σ_(gl)represents the surface tension of the fluid with respect to the air(millijoules/m²), g is the gravitational constant (m/s²), r is theradius of the capillary tube (m), and ρ is the fluid density (kg/n³).

Planar micro structures have been developed in which a number of groovesor channels are made. Typically, such a planar structure is produced byetching grooves in a semiconductor substrate, such as a silicon wafer,and then covering the etched surface by a cover plate to complete thechannels. Such structures are, however, rather time consuming andexpensive to produce.

Further, when such structures need to be customized, e.g. by theaddition of chemical reagents, the functionalization of surfaces etc.,these steps often need to be performed by somebody other than theproducer of the micro structure. In practice, the micro structures aremanufactured at one location and shipped to another facility, e.g. forthe addition of reagents, whereupon they often need to be returned tothe manufacturer for closing and sealing. One aim of the presentinvention is therefore to make available a micro structure offeringgreater flexibility and ease, in particular with regard to thepost-production customization.

Systems used at present utilize external means, e.g. gravity,centrifugal force (spinning of disk elements with micro channels on thesurface), or pressure to impose transport of liquids in channels. Also,electric fields can be used to impose transport of dissolved chargedspecies in micro systems. To this end, external auxiliary equipment isemployed, such as a motor to generate the spinning of a disk, pumps tocreate pressure, electrodes and power supplies to apply electric fields,etc. Such equipment is costly and sometimes rather complex. Furthermore,in certain cases the forces involved in the above mentioned methodscould have detrimental effects on sensitive substances. Another aim ofthe invention is therefore to make available a micro structure withbuilt-in functionality, removing or reducing the need of external meansto impose liquid transport.

PRIOR ART

EP 1 120 164 describes the use of a plurality of micro structures in acapillary pathway, said pathway having at least one curved portion, thepathway comprising a base, an inner wall defined by a first radius froma center point and defined by a second radius greater than the firstradius, the inner wall and outer wall being fixed to the base anddefining the lateral boundaries of the capillary pathway, and a lidextending at least from the inner wall to the outer wall covering thecapillary pathway. It becomes clear that the micro structures themselvesdo not form a capillary pathway, but only influence the flow as thefluid travels around curved portions in said capillary pathway, the flowbeing somewhat slower near the inner wall of a curved portion than nearthe outer wall.

U.S. Pat. No. 5,885,527, cited in the above EP 1 120 164 describes assaydevices including micro structures forming reaction barriers. Thesereaction barriers are formed by corrugated or other otherwise patternedsurfaces, having grooves which together with a cover or top member formnarrow channels between different chambers in said device. It is clearfrom the description and figures, that the capillary channels are indeedformed only when a top member is placed on a bottom member a capillarydistance apart. Further, the top and bottom members may be married, thevarious chambers sealed and the capillaries formed by a number oftechniques, including but not limited to, gluing, welding by ultrasound,riveting and the like.

U.S. Pat. No. 5,837,115 discloses a sorting apparatus and method forfractionating and simultaneously viewing individual micro structures,such as cells, viruses, macro molecules or minute particles in a fluidmedium. The aim of this invention is to replace agarose gels and othertraditionally used fractionation media with a lattice structure withuniform distribution, size and shape of the hindered environment. Thehindered environment may be formed by posts, bunkers, v-shaped orcup-shaped structures, forming sifting means for the cells, viruses,etc. under study. This structure is covered by ceiling means positionedover the lattice causing the migration of microstructures in essentiallya single layer through the sifting means exclusively. The inventionaccording to U.S. Pat. No. 5,837,115 does not seem to consider capillaryforces, but instead suggests the provision of electrodes for generatingan electric field over the lattice structure.

It is widely known to use channels filled with porous material, asexemplified in U.S. Pat. No. 5,540,888, said porous material havingsub-channels divided by slots, liquid impermeable separating portions,etc., and containing reagent regions and sample application regions.Filter paper is often the material of choice, and there exists a widerange of technologies for applying reagents to this material.

It remains to make available micro fluidic structures integrated in asupport which is suitable for mass production, and in a configurationwhich makes the micro fluidic device, comprising said structures, easilyhandled in the down-stream production process, and in particular in thecustomization of the device.

In view of the drawbacks of having to use relatively complex externalequipment for influencing liquid flow, which in addition could damagethe samples, it would be both desirable and advantageous to enable“automatic” transport of sensitive substances without use of complexdevices and without subjecting the substances to the risk of beingaltered, e.g. denaturated or otherwise damaged.

One aim of the present invention is therefore to provide a micro-fluidicstructure, i.e., a geometric micro structure defining a liquid flowsystem, suitable for capillary transport of liquids and which isinexpensive to produce, optionally permitting a disposable type product,optionally having branched flow channels, optionally exhibiting localsurface characteristics, and providing great freedom in choice ofmaterial, e.g. with regard to surface, optical and electric properties.

Further aims underlying the invention, as well as the advantagesassociated with the inventive solution and its embodiments, will becomeapparent from the following description and examples, together with theattached claims and drawings.

SUMMARY OF THE INVENTION

The above aims are achieved with the present invention by employing thephenomenon of capillary action to drive liquid flow through open microstructures provided on solid substrates. In its widest scope, the microfluidic structure according to the present invention comprises variousforms of geometric micro structures defining the desired liquid flowsystem.

The present invention in its broadest aspect provides a flow pathconsisting of a multitude of micro structures inducing and/orfacilitating the flow of fluids through said flow path, as well asmethods where this flow path is used.

The invention in particular provides a micro fluidic system comprising asubstrate, and, provided on said substrate, at least one flow path andfunctional means in which liquid samples can be treated by desiredprocedures, wherein said at least one flow path is/are laid out to forma pattern for the transport of liquid samples to, through and from saidfunctional means; wherein the flow path consists of a plurality of microposts protruding upwards from said substrate; wherein the spacingbetween the micro posts is such as to induce a capillary action in aliquid sample applied anywhere to said flow path, so as to force saidliquid to move from where said liquid sample was applied.

The functional means may comprise one or more of chemical reactors,separation means, heating means, means for irradiation withelectromagnetic radiation, magnetic means for trapping magneticcomponents of said liquid within said functional means, electrodes forapplying voltage to the liquid over a selected region, or any otherdevice or means for chemically, biologically or physically treatedliquid samples located within said functional means.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in closer detail in thefollowing description and non-limiting examples, with reference to theattached claims and drawings, in which:

FIG. 1 is a SEM microphotograph of a portion of a structure according tothe invention;

FIG. 2 through 6 show cross sections of the capillary structureaccording different embodiments of the present invention;

FIG. 7 shows a perspective view of one embodiment of a flow path wherethe capillary structure of micro posts having a circular cross section;with no walls nor cover which would significantly contribute to thecapillary action, according to the present invention;

FIG. 8 illustrates one embodiment of a flow path (1.1) according to thepresent invention comprising zones with capillary structures or microposts having different cross sections and different dimensions inadjacent zones;

FIG. 9 shows schematically in cross section perpendicular to the planeof the device, the use of a bibulous material for maintain or enhancingflow in a flow path according to the present invention;

FIG. 10 shows schematically how a flow path according to the presentinvention can be divided into zones, separated by “barriers” preventingthe capillary flow, and means for resuming or re-starting the flow;

FIG. 11 shows schematically a cross section of a device having microstructures forming a flow path on a surface, and a separate lid, notsignificantly contributing to the capillary flow;

FIG. 12 shows schematically a partial exploded view of a deviceaccording to the invention, and as illustrated in FIG. 11, where a dropof liquid, e.g. a sample, is being added with a pipette through a holein said separate lid;

FIG. 13 shows schematically an embodiment where microposts of differentsize and/or functionality form a discontinuous gradient; and

FIG. 14 shows a perspective view of one embodiment of a flow path wherethe capillary structure is formed of micro posts in a groove of asubstrate, according to embodiments of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described, it is to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims and equivalents thereof

The following terms will be used:

The prefix “micro” as in micro fluidic, micro structure, etc. is used todefine a device or a process which comprises or involves at least onefeature having a length, width or height normally expressed inmicrometers (μm, 1×10⁻⁶ m).

The prefix “nano” is here used in its generally accepted meaning, as innanometer (nm, 1×10⁻⁹ m).

The expression “passive” as used in e.g., “passive control” or “passivefluid dynamics: this term refers for the purposes of this invention to acontrol that is not influenced by actions taken during a process that isto be carried out, but rather the control is determined by fixed systemparameters, which are design dependent. The passive control is generatedby using the natural capillary forces that exist on a micro scale.

The term “open” in this context means that the flow paths defined by themicro structures are accessible from above, and have no cover or lidwhich takes part in creating the capillary flow. The above definitiondoes however not rule out that a secondary cover or lid, at a distancefrom the micro structure, can be provided.

The term “hydrophilic groups” refers to substrates and substances havingpolar and/or charged groups, such as hydroxyl, carboxyl, amino,sulphonate, thiol, aldehyde, etc.

The term “hydrophobic structure” refers to substrates and substanceshaving non polar structures.

The term “chemically reactive groups” refers to all organic andinorganic groups used in covalent coupling of molecules to solid facesand known to persons skilled in the art, such a hydroxyl, carboxyl,amino, sulphonate, thiol, aldehyde, etc.

The term “biological affinity” refers to substances having specificbinding of a substance or a defined group of related substances.Exemplary substances are antibodies, antigens, haptens, biotin, avidin,lectin, sugar, nucleic acids, hormones and their receptors.

The invention in its broadest aspect provides a multi-element capillarystructure adapted to facilitate and/or effect the capillary flow ofliquids along said structure in open systems by using micro-structuresprovided on or in said structure. Advantage is taken of the surfaceeffects between a fluid and the surfaces contacting the fluid. Thesesurface effects come into play at the micro scale.

In particular there is provided devices having a structure comprising atleast one liquid flow path, optionally connecting different processingcompartments within said structure for carrying out a number ofdifferent unit operations. Examples of such processing compartments,elements and/or devices are chemical reaction compartments, incubationcompartments, wash compartments or elements, flow control elements,measurement elements, time gates, separation means, heating means, meansfor irradiation with electromagnetic radiation, magnetic means fortrapping magnetic components of said liquid within said functionalmeans, electrodes for applying voltage to the liquid over a selectedregion, detectors for detecting physical or chemical properties e.g.,temperature, pH, viscosity, absorbance, etc., or any other device ormeans for chemically, biologically or physically treating a liquidsample or reaction mixture located within or passing through saidcompartment, element and/or device.

In order to clarify the principle behind the inventive concept, thefollowing illustration is given:

Consider a simple example of the effect of surface forces, such asdemonstrated when water is drawn into a glass capillary without anyoutside pressure being applied. This is caused by the surface tensionbetween the water and the glass surface, which pulls water into thecapillary. The narrower the capillary, the greater the effect of theforce that pulls the water into the capillary. This is often referred toas the capillary force.

One physical parameter that characterizes the magnitude of the capillaryforce is the contact angle between the water and the surroundingmaterial. For contact angles less than 90°, the material, e.g., glass,is considered to be hydrophilic and water is drawn up into the capillaryspace. When the material has a contact angle greater than 90° it isconsidered hydrophobic. In the hydrophobic case, pressure is required toforce water into the space. The narrower the capillary, the greater theforce that is required. However, in both cases once water has beenintroduced into the capillary, the flow rates of the water depend moreon pressure gradients and friction and less on whether the material ishydrophobic or hydrophilic.

In order to achieve the desired function of the micro structures formingthe flow paths of the present invention, the contact area between liquidand the surface of the solid material is maximized, whereby thecapillary force increases so that a fluid flow within or along the flowpath according to the invention is spontaneously induced and maintainedover a desired period of time.

This process represents what can be referred to as “passive fluiddynamics”. The present inventors have found that it is advantageous touse such passive fluid dynamics to control and drive the flow of fluidin open micro channels or structures on a surface. For example, thepassive nature of the transport mechanism according to the inventionmakes it direction independent, as compared to a system on a spinningdisk where mainly a radial transport is possible. Transport byapplication of an electric field is mainly bi-directional at best.Capillary flow can be induced in any direction, provided the pattern ofmicro structures forming the flow paths is designed properly. A personskilled in the field of designing micro structures and fluid flowchannels on such structures will be able to apply the teaching of theinvention without undue experimentation.

FIG. 1 shows a SEM microphotograph of an example of a micro fluidicstructure embodying the inventive concept. It is evident from thepicture that the micro posts have an identical shape, a regular form andare evenly spaced over the support structure. Also, the surface betweenthe micro posts is even. A skilled person will recognize that the microposts shown in the SEM microphotograph have a high aspect ratio. In thisexample, the micro posts were about 100 μm high, had a diameter of 20 μmand a center-to-center distance of 30 μm. This accounts for an aspectratio of 1:5. It is generally held that an aspect ratio >1:2 is a highaspect ratio.

FIGS. 2 through 6 show a number of different cross sections of the microstructures forming the fluid flow path according to the presentinvention. The micro structures or micro posts can have a cross sectionwhich is one of circular, elliptical, rhombic, triangular, square,rectangular, heptagonal, hexagonal, etc. or a combination thereof. Thecross section can also be any fraction of the above forms, such a ashalf-circle, a crescent, U-shaped, X-shaped, etc. as long as thedimension and center-to-center distance of the individualmicrostructures is such, that capillary flow is induced without theprovision of any lid or cover, limiting the flow path.

FIG. 7 shows schematically a flow path consisting of a multitude ofcircular micro posts 1 on a surface 2. The micro posts can naturallyhave any cross section, height and center-to-center distance, as long asthe parameters are chosen such that capillary flow is induced. Thedirection of the capillary flow is indicated by the black arrow. In FIG.7, the support 2 is schematically indicated as having a thicknesscomparable to the height of the micro posts. While this is not ruledout, the most frequently encountered supports will be considerablythicker. FIG. 7 is thus only a schematic illustration.

A device according to the invention may comprise a support and at leastone flow path consisting of micro structures as shown in FIG. 7.However, for most practical applications, a multitude of flow pathsforming a channel system is required. The individual channels connectdifferent functional regions, means or devices, such as reactionchambers, separation media etc.

When the properties of the columns and their properties, such as theirmaterial, their shape and/or distance, and optionally also theproperties of the substrate, are selected properly with dueconsideration taken to the liquid to be transported, it will becomepossible to create a capillary flow through said structure, in thedirection of the arrow if a liquid sample is placed at the end where thearrow points, without any significant leakage out from the structure andonto the surrounding substrate surface.

Thus, the basic structure embodying the inventive concept is a substratehaving at least one flow path provided in or on its surface. This flowpath or channel is formed by column like micro structures or microposts, protruding from the surface of said support. The characteristicfeature of the flow path and the micro posts therein is that thedimensions of said posts and the distance between said posts areselected such that capillary flow of liquids can be maintained therein.In particular, the distance between said columns is in the range of0.1-1000 μm, preferably 1-100 μm. The columns are preferably higher than1 μm, more preferably higher than 10 μm. Most preferably said microposts have a high aspect ratio; that is, a width to height ratio greaterthan 1:2.

It is understood that the micro posts can be either positioned within asecondary structure on the surface, with reference to FIG. 14, such as agroove (1.6) or a depressed area, or directly on the surface, protrudingtherefrom. When the micro posts are in a secondary structure, such as agroove in a substrate (2), the flow path will have a bottom (1.7)located beneath the general substrate surface, and more or less verticalside walls, together with the bottom forming a channel. The capillaryaction inducing and/or maintaining the flow however, is causedessentially by the interaction between the liquid and the micro posts.

However, according to a preferred embodiment, the micro posts or columnsare laid out as regions, preferably elongated, of upstanding columns,without any delimiting side walls. All functions and features that havebeen or will be discussed herein with reference to ordinary channels,are equally applicable to this type of structure, which thus is fullywithin the scope of the inventive concept as defined in the claims.

According to a further embodiment of the present invention, the flowpath is subdivided into zones wherein the columns can have differentcolumn heights, diameters, geometry and/or different column density,i.e., number of micro-posts per unit area. FIG. 8 is a schematicalillustration of this embodiment. A plurality of micro-posts 1 providedin close proximity in adjacent groups are indicated 8, 9 and 10 and thedifferences shown as different size, shape and spacing are provided onlyfor the sake of illustration. Such groups can form an array, havingdesired functionality. Preferably said groups form a gradient, which canbe continuous or discontinuous, preferably continuous.

In the embodiment shown in FIG. 8, a first more dense zone 8 isprovided, where the micro structures have a smaller diameter and smallerdistance. Such zone can act as a sieve or “fence” preventing largerparticles, e.g., cells from passing. Next, there is a zone 9 with postshaving relatively large spacing between them. This can serve totemporarily decrease the time for a liquid-solid face interaction in adesired region, e.g. if it is desired that the sample be exposed to somesurface bound moiety for a specified time, in order for a particularreaction to proceed to a reasonable completion etc. After this lowvelocity region there is provided a zone 10 of larger micro posts(squares in the shown example) having fairly narrow passages betweenthem. After this region, a second zone 9 similar to that is provided.Based on the information given in this description and the examples, askilled person can design various combinations, according to the desiredpurpose.

According to another embodiment of the invention, the capillary flowpath or paths has/have integrated surfaces or zones composed of bibulousmaterial capable of capillary transport. This is schematicallyillustrated in FIG. 9 which shows a simple application of this concept.In this case, there is a closed channel structure comprising a pluralityof micro-posts 1 filling the channel. The structure has a bottomsubstrate 2 and a cover 6, the substrate also forming side walls (notshown), and having an input aperture or hole 7, an optional furtheraperture or hole 8, and an exit aperture (not shown). If a drop 9 ofliquid is applied to the input aperture 7, a capillary flow willimmediately begin, and will continue to draw liquid from the drop untilthe flow reaches the exit, where no further capillary action will occur.However, if a bibulous material such as pad 10 of filter paper or thelike of sufficient size, is applied at, within or in contact with theexit aperture, this material will by its capability of drawing liquidact as a “flow sink”, i.e. it will absorb the liquid that exits from thechannel, thereby enabling an essentially continuous flow through thechannel.

Another embodiment having a flow sink, is that of the closed channelending in an open region or zone of micro posts, said region having arelatively large area as compared to the channel. This large region willact as a flow sink in the same sense as the bibulous material discussedabove. If e.g., heating means is provided to heat this region,evaporation of the liquid can be induced, thereby creating a sink thatin principle could be maintained indefinitely. Evaporation of the liquidwill also make possible the capture of components present in the flow,at a desired location and at a desired time in the device or processemploying such device.

FIG. 10 shows schematically another embodiment and application of theflow path structures according to the invention, the flow pathcomprising a plurality of consecutive micro fluidic structures,interrupted by zones without such structures, i.e., a small space ordiscontinuity 11 and 12, said discontinuities acting as capillarybarriers, preventing liquid from being transported across the zoneswithout assistance. The direction of the liquid flow is illustrated withthe large horizontal arrow.

The discontinuities can be bridged or assisted transport can be broughtabout e.g., by applying a pressure pulse, whereby the capillary barriersprovided by the voids are broken. The means for creating the pulse canbe implemented by providing a very small channel 13 and 14 opening intothe spacing, and for example momentarily applying sub-pressure(indicated with vertical arrows) in said channels. At the other end ofthe channel some means for providing a slight sub-pressure can beprovided, whereby liquid from the regions on each side of thediscontinuity will be forced into it, and when the gap is filled,capillary flow will again be resumed. This requires a closed system. Inan open system (and in a closed), the small channel can be used tointroduce liquid to fill the gap, thereby restoring a flow connection soas to resume the capillary flow.

FIG. 11 shows a schematic partial view of a device according to theinvention, where micro posts 1 are provided on a substrate 2, saidsubstrate having larger protrusions 16 for carrying a cover or lid 15,said protrusions defining a distance between the surface, the substrateand the lid considerably larger than the height of the micro posts, andsuch that no capillary interaction between the substrate and the lid canarise. The same applies to an alternative embodiment where the microposts are located in grooves or depressions in the surface. The cover orlid 15 may further have apertures or holes 17, said holes preferablyindication points for adding a sample or a reagent, for reading a resultor for following the advancement of the reaction/reactions taking placeon the substrate.

According to a preferred embodiment, the cover or lid is attached to theprotrusions 16 only after the surface of the substrate has beenfunctionalized or customized, i.e. after the addition of the necessaryreagents and/or functionalities.

FIG. 12 shows schematically an exploded view of the embodiment of FIG.11, where a pipette tip 18 is shown depositing a drop of a liquidthrough an opening 17 on a flow path according to the invention.

FIG. 13 illustrates an embodiment where groups of micro structureswithin a continuous flow path (1.1) form a gradient with respect to atleast one property, e.g., the shape, size, center-to-center distance ora functional/chemical property. In the figure, the flow path comprises adiscontinuous gradient with respect to the size and center-to-centerdistance of the micro structures, represented by the groups A, B, C andD. A gradient like this can function to delay the passage of biologicalor chemical entities, such as particles, cells, organelles, macromolecules or the like, in a desired manner, or separate such entities.Entities captured in zone A are illustrated by the shape “a”, entitiescaptured in zone B with “b” and so on. The shape “e” illustratesentities that pass unhindered through the gradient.

In further embodiments, the flow paths can comprise integrated surfacesor zones containing a number of different functional elements or devicesfor performing a number of different operations on the media locatedwithin or in association to the flow paths. Examples of such functionalelements or devices are electrodes (1.2) and/or other means ofelectrical manipulation of liquids and reagents. The electricalmanipulation can e.g. comprises oxidation/reduction of species. Otherrepresentative examples of functional devices are optics and other meansto manipulate light e.g., for the purpose of measuring concentrations byabsorbance, inducing conformational changes by light irradiation.

Magnets or means for detection of magnetic substances (1.3) can also bearranged in or around the flow paths. Thereby, magnetic particles can betrapped and retained at desired locations in the structure, and themagnetic property of the particles can thereby be used as a marker orindicator of successful transport to a certain point in the system.Furthermore, magnetic particles can be coated with substances withbiological affinity and used in different kinds of assays.

Furthermore, means for the manipulating of temperature with means forheating and/or cooling (1.4) can be provided in selected locations in aflow path according to the invention. Thereby a number of interestingfunctions can be performed such as, chemical reactions, incubation,thermal curing, PCR reactions, evaporations, drying, etc.

Moreover, means for applying energy (1.5) may apply energy to one ormore selected segments so as to induce a forced transport across theflow stop. The means for applying energy may be a pressure pulsegenerator, an ultrasound generator, or an electromagnetic radiationmeans, for example.

Within the flow path structures according to the invention there can beprovided particles within or in association to the flow paths, at leastin one of the zones containing micro structures such as micro posts orcolumns, where there is subdivision between zones with and without thesestructures. The particles can thereby be bound by physical forces tosaid substrate, or chemically bound, e.g. covalently bound to saidsubstrate. Alternatively, said particles can be mechanically trappedwithin the zone (zones) containing said structures.

According to a preferred embodiment, the substrate has reactivesubstances attached to its surface. Such reactive substances are used inconnection with detection of substances of chemical or biologicalorigin. The reactive substances are used in connection withimmobilization of substances of biological as well as non-biologicalorigin, i.e., they are provided as sites to which the substances to beimmobilized can react so as to become bound to the substrate.

Another application of a reaction substance provided on the substrate isthe use thereof in connection with separation of substances ofbiological as well as non-biological origin, where the substance can beselectively reactive with a species that one wishes to separate fromanother in a mixture.

The surfaces of a channel in a structure according to the invention canbe modified by chemical or physical means. Such a modified surface canthereby be used in connection with detection of substances of biologicaland chemical origin. A modified surface of this kind can be used inconnection with immobilization of substances of biological, as well asnon-biological origin. It can also be used in connection with separationof substances of biological, as well as non-biological origin.

Still another application of the flow path or channel structureaccording to the invention, is that the flow paths are used as means formulti spot detection, whereby said paths optionally have particlesprovided therein, said particles having chemically reactive groups orsubstances with bio-affinity bound in the micro spot area.

Other suitable applications of flow path structures according to theinvention is the measurements of the amount of an analyte in abiological sample, e.g., in blood, serum, plasma, urine, cerebral spinalfluid, tears, amniotic fluid, semen or saliva, the measurementspreferably being based on specific biological interactions.

A specific application is that said analyte is measured by immunologicalmeans.

The analyte can also be detected by specific interactions using poly- oroligonucleotides, preferably single stranded nucleic acids or aptameres.

The flow path structures according to the invention can also be used forthe separation of cells, and for the screening of synthetic orbiological libraries.

As discussed above, the notion of a “channel” for the purposes of thepresent invention goes beyond the ordinary concept of a channel, byallowing entirely open structures having no physical delimitationsexcept for a bottom substrate on which the micro post or columnstructures are provided.

However, when the channel or flow path comprises a groove (i.e., havinga bottom and side walls), there are two options: i) to leave the channelopen upwards, and ii) to place a top cover so as to make a closedsystem.

These two different embodiments have certain merits for differentapplications. In cases where it is desirable to manipulate the liquidthat is transported and/or the substances transported by said liquid, itmay sometimes be more convenient to have unrestricted access from above,such as for the purpose of adding reagents at desired points in thestructure, to apply direct heat, or to perform other manipulations atmore or less arbitrarily points.

Certain systems may be very sensitive to oxygen, and therefore it may beabsolutely necessary to exclude the sample liquid from atmosphericexposure. If the micro-fluidic structure is covered, there can beprovided access openings in said cover for enabling introduction ofe.g., reagents, gas, liquids, or samples into said structure. Suchaccess openings can also be used for connecting external equipment,e.g., via suitable tubing or the like.

However, both these embodiments are within the inventive concept.

Manufacturing of such microstructures could in its simplest form be doneby direct curing of a photosensitive mono- or pre-polymer deposited on asubstrate, employing a mask through which light is irradiated toinitiate curing, and therefore rinsing away the un-cured areas (thickfilm photo-resist process).

Another straightforward method is through replication of an originalinto a polymer. The original could be manufactured in silicon through aDRIE-process (Deep Reactive Ion Etch) where high aspect ratio structurescould be produced. Other ways of producing such originals could forinstance be through laser processing, electro discharge methods, FreeForm Manufacturing (FFM), electrochemical or chemical etching, gas phaseetching, mechanical processing, thick film photoresist processes orcombinations thereof, of or on a substrate of, for instance, silicon,glass, quartz, ceramic, metal or plastic material, e.g., PMMA or Teflon.

The most straight forward method of replication would be casting of amono- or pre-polymer over an original with the desired negative shape.Other ways of producing the polymer replicas could involve injectionmolding or embossing of thermoplastics or thermoset materials.

If the original in some aspects are not withstanding the replicationprocess, an intermediate replica in a suitable material (a stamper)could first be produced from the original. Examples of such a stamperprocess could be to first deposit a conducting layer on top of theoriginal and thereafter through electroplating form a negative from theoriginal. Certain plating materials, such as Nickel, lend themselvesalso to be repeated and non-destructive production of copies of thestamper. This gives the possibility to both change polarity fromnegative or positive, as well as producing series of identical stampersfor large volume production of replicas. Other examples of stampermanufacturing could be in a well chosen polymer given the negative shapeof the original in a casting, embossing or injection molding process.The same possibility of repeatedly and non-destructively making copiesof the stamper could also be true for polymer stampers.

As mentioned above the micro-fluidic structure of the invention may, ofcourse, be designed for a plurality of micro-fluidic purposes. Amongthose are e.g., capillary chromatography, ion-exchange chromatography,hydrophobic interaction chromatography, immunoassays, hybridizationassays and other molecular biology assays, micro reaction cavityprocedures, miniaturized liquid communication units, biosensor flowcells, etc. Reaction cavities constructed in accordance with theinvention may, for example, be used for various forms of solid phasesynthesis, such as peptide or oligonucleotide synthesis, PCR, DNA solidphase sequencing reactions, sample treatment and detection, just tomention a few.

The micro fluidic structures according to the invention can be made indifferent ways. One convenient method is outlined above, but it is alsopossible to make the structures from separate parts which are assembledafter column formation has taken place in a suitable substrate.

In the following, the invention will be illustrated by specificnon-limiting examples.

EXAMPLES Example 1 Flow in Open Channels with Columns Made of Silicon

Flow channels were produced by etching silicon wafers by a standardmethod well known to a person skilled in the art. The resulting siliconchips had a length of 25 mm and a width of 5 mm. The area covered bycolumns was 10 mm long and 4 mm wide. The columns had a height of 100 μmand a diameter of 20 μm, the center-to-center distance being 30 μm.

Capillary flow was tested with purified water, buffer and blood plasma.A wicking membrane (Whatman WF 1.5) was placed a few mm in at thedistance end of the chip to facilitate the liquid flow.

8 μl of water added to the structure took between 60 and 90 seconds toflow across the structure provided with columns. A similar flow speedwas measured with a buffer containing 50 mmole/l sodium phosphate, 6%bovine serum albumin, 0.2% Tween 20, pH 7.5. Blood plasma was slightlyfaster than water and buffer.

Example 2 Flow in Open Channels with Columns Made of Epoxy Plastic

Flow channels were produced by first etching silicon wafers by astandard method well known to a person skilled in the art. A thin layerof epoxy was applied uniformly to the silicon wafer. The resulting epoxycovered chips had a length of 25 mm and a width of 5 mm. The areacovered by columns was 10 mm long and 4 mm wide. The columns had aheight of approximately 90 μm and a diameter of approximately 20 μm, thecenter-to-center distance being close to 30 μm.

Capillary flow was tested with purified water and buffer. A wickingmembrane (Whatman WF 1.5) was placed a few mm in from the distal end ofthe chip to facilitate the liquid flow. The chip was pretreated (onehour, room temperature) with a buffer containing 50 mmole/l sodiumphosphate, 6% bovine serum albumin, 0.2% Tween 20, pH 7.5 prior toaddition of water or buffer. 8 μl of water added to zone 1 took about 90seconds to flow through the zone with columns. Similar flow speed wasfound with a buffer containing 50 mmole/l sodium phosphate, 6% bovineserum albumin, 0.2% Tween, 20, pH 7.5.

The above examples demonstrate that it is possible to create open flowpaths consisting of micro structures and that these function, i.e., thatthe necessary capillary forces are created and that liquid transporttakes place.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

1. A method for measuring the amount of analyte in a biological sample,the method comprising the steps of: providing a micro fluidic system,the system comprising: a substrate having a non-porous surface; aplurality of microposts protruding from the non-porous surface of thesubstrate, said microposts defining at least one open flow path in whichliquid samples can be treated by desired procedures, wherein the atleast one flow path is laid out for transport of liquid samples, the atleast one flow path extending in a lateral direction which is transverseto the protruding microposts, wherein cross sections of the micropostsand the center to center spacing between each of the micropostsspontaneously induces a passive capillary action in a liquid sampleapplied to the at least one flow path, so as to force the liquid to movelaterally away from where the liquid sample was applied, the micropostsfurther including antibodies having an affinity for the analyte in thesample; a sample receiving area disposed prior to the at least one flowpath and fluidly connected therewith; and a sample collecting sinkdisposed after the at least one flow path on the substrate and fluidlyconnected with the at least one flow path, the sample collecting sinkhaving an area which is larger than the area of the at least one flowpath extending from the sample receiving area, the sink having aplurality of microposts protruding from the substrate surface, the sinkmicroposts having cross sections and center to center spacing betweeneach of said microposts that further induce capillary flow from the atleast one flow path to the sample collecting sink; applying thebiological sample containing analyte to the sample receiving area;flowing the sample through the at least one flow path, whereby theanalyte in the sample is captured by the antibodies; and measuring theamount of analyte by immunological means.
 2. The method according toclaim 1, including the step of providing functional means comprising oneor more of chemical reactors, separation means, heating means, means forirradiation with electromagnetic radiation, magnetic means for trappingmagnetic components of said liquid within said functional means,electrodes for applying voltage to the liquid over a selected region, orany other device or means for chemically, biologically or physicallytreating liquid samples.
 3. The method according to claim 1, wherein thesubstrate is provided with grooves having a bottom surface and sidewalls, and wherein the at least one flow path includes the micro postsprotruding from the bottom surface of the grooves.
 4. The methodaccording to claim 3, including the step of providing a top or lidcovering the at least one flow path, wherein the top or lid does notsignificantly contribute to the capillary action in said at least oneflow path.
 5. The method according to claim 4, including the step ofproviding the lid or top with access openings for enabling introductionof reagents, gas, liquids, samples into the at least one flow path. 6.The method according to claim 1, including the step of grouping theprotruding microposts along the at least one flow path in adjacentsegments so as to provide a spacing or discontinuity between suchsegments, the discontinuity having a finite distance, which is largeenough to prevent capillary flow between the segments, thereby providinga flow stop.
 7. The method according to claim 6, including the step ofproviding means for applying energy to the one or more selected segmentsso as to induce a forced transport across the flow stop.
 8. The methodaccording to claim 7, wherein the energy applying means is selected fromat least one of the group consisting essentially of a pressure pulsegenerator, ultrasound generator, and an electromagnetic radiation means.9. The method according to claim 6, comprising the step of providingmeans for applying liquid to the discontinuity in order to provide abridge across said discontinuity so as to induce a flow there across.10. The method according to claim 1, including the step of providing achemical, biologic or physical functionality to the surfaces of themicroposts wherein the functionality is defined by the microposts havingat least one of the group consisting essentially of chemically reactivegroups, substances with biological affinity, hydrophilic groups,hydrophobic structures, and positively and/or negatively charged groupson their surfaces.
 11. The method according to claim 10, wherein thestep of providing a functionality includes the step of providing thefunctionality to the micro posts over the entire at least one flow pathor limiting the functionality to a discrete region or portion of the atleast one flow path.
 12. The method according to claim 11, wherein saidproperties are selected from the micro post diameter, height, shape,cross section, surface coating, number of micro-posts per unit area,wetting behavior of the micro-post surface, or a combination thereof.13. The method according to claim 1, including the step of providingparticles within the at least one flow path.
 14. The method according toclaim 13, including the step of chemically or physically bondingparticles to the substrate, or mechanically trapping the particleswithin a region comprising a plurality of the protruding microposts. 15.The method according to claim 1, including the step of providing the atleast one flow path with integrated zones or delimited surfacescontaining electrodes or other means for electrical manipulation ofliquids and/or reagents.
 16. The method according to claim 1, includingthe step of providing the at least one flow path with integrated zonesor delimited surfaces, the zones or surfaces containing at least one ofoptical elements or other means for transmitting, focusing, reflectingor absorbing light, magnetic functionalities or means for manipulationand/or detection of magnetic substances, and means for the regulation ofthe temperature in the zone, e.g., heating or cooling said zone.